Previous work has shown that nonmuscle myosin heavy chain II-B (NMHC II-B) ablated mice develop an unusual form of myocyte enlargement which appears during embryonic development and involves both atria and ventricles. Cardiac myocytes differ from the non-myocytes in the heart (fibroblasts, endothelial and smooth muscle cells, etc.) in not containing NMHC II-A. Recently, we have demonstrated that a third form of nonmuscle myosin II, NMHC II-C, exists in human and murine cardiac myocytes using immunofluorescence. Thus, cardiac myocytes ablated for NMHC II-B might be expected to show a defect in cytokinesis, since both NMHC II-A and II-B have been shown to participate in this process. To study this possibility, we quantitated the number of cardiac myocytes in B-/B- mouse hearts compared to control hearts (B+/B-, B+/B+) at E14. We found that the number of cardiac myocytes per heart section was decreased by more than 50% in the B-/B- heart, but that the number of non-myocytes, all of which contain NMHC II-A, was the same for both B-/B- and control hearts. As further evidence for a defect in cytokinesis, but not karyokinesis, we quantitated the extent of binucleation and found that, at E12.5, 23% of the B-/B- cardiac myocytes were binucleated compared to 1% of the cardiac myocytes in control hearts. There was no evidence for binucleation in the non-myocytes of the B-/B- hearts. We compared the extent of myocyte proliferation by measuring the BrdU labeling index (% myocytes labeled with BrdU) at E14 and found that it was significantly reduced for B-/B- cardiac myocytes, but not for B-/B- non-myocytes in the heart nor for other organs. We interpret these data as evidence for a previously undescribed cause of cardiac myocyte enlargement, in which cytokinesis, but not karyokinesis, is partially defective, thereby impairing myocyte proliferation during embryonic development. We attribute the partial, rather than complete, failure of cytokinesis to the presence of NMHC II-C. However, NMHC II-C alone does not appear to be sufficient for normal cytokinesis. In contrast, the cardiac myocytes of hypomorphic mice expressing only 7% of the normal amount of NMHC II-B in the heart at E12 showed no evidence of binucleation and hypertrophy, consistent with the restoration of normal cytokinesis. Interestingly, we also found that hypomorphic homozygous mice harboring the single NMHC II-B mutation R709C also have a decreased number of cardiac myocytes, many of which show evidence for binucleation and enlargement.